Journal: Cell Reports Methods

Article Title: Generation of T cell responses against broad KRAS hotspot neoantigens for cell therapy or TCR discovery

doi: 10.1016/j.crmeth.2025.101049

Figure Lengend Snippet: Enrichment of antigen-specific T cells (A) Representative flow-cytometry plots demonstrating the upregulation of activation markers on antigen-specific CD8 + cells (red) compared to bystander cells (gray). Red numbers: % of CD8 + cells. (B) Aggregate data from six independent experiments using either 4-1BB (left) or CD69 (right) as enrichment marker. Note increase in frequency or de novo detection of antigen-specific cells. Colors denote different KRAS neoantigens. (C and D) Enrichment of antigen-specific CD4 + cells with either 4-1BB- or CD69-based enrichment compared to a non-enriched condition. (C) Representative flow-cytometry plots from one healthy donor identifying mKRAS-specific CD4 + T cells via the delta IFNγ or TNF-α expression with or without an overnight peptide stimulation. (D) Responses across three donors (4-1BB in dark green, CD69 in green). Horizontal bars indicate the mean. p values per one-way ANOVA with Dunnett’s correction for multiple comparisons: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (E) First four runs of the large-scale enrichment process. Cells were expanded post enrichment and antigen-specific CD8 + T cell frequency was determined pre-enrichment (day 13), post enrichment (day 14 or day 16), and post expansion (day 26). Dotted line indicates target frequency. (F and G) In vitro cytotoxicity using a representative DP. DP was co-cultured with GFP-labeled target cells. (F) A375 melanoma cells transduced to express HLA-A∗11:01 at an approximate antigen-specific effector to target (E:T) ratio of 5:1. Target cells were either loaded with WT KRAS peptide (purple) or G12V neoantigen peptide (blue). (G) SW620 colorectal adenocarcinoma cells, which endogenously express KRAS G12V, transduced to express HLA-A∗11:01 at an approximate antigen-specific E:T ratio of 2.5:1 (blue). Data points represent the mean of GFP-labeled target cell area from technical duplicates (F) or triplicates (G) ± SD. p values per two-way ANOVA: ∗∗∗∗ p < 0.0001. See also .

Article Snippet: CD69 + Microbeads , Miltenyi , Cat#130-092-355.

Techniques: Flow Cytometry, Activation Assay, Marker, Immunopeptidomics, Expressing, In Vitro, Cell Culture, Labeling

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Blended projections of T cells (red) clustered around a conduit (white)-associated VACV-infected cell (green) near a high endothelial venule (HEV, indicated in picture). Arrows point to conduits. Scalebar = 20 μm. Area in circle is magnified on the right. Scalebar = 5 μm. ERTR7= white, VACV-infected cells = green, OT-I CD8 + T cells = red, B220 = blue. Results are representative of 30 LN sections taken from 5 experiments. b ) OT-I CD8 + T cell activation as determined by flow cytometry of single-cell suspensions of popliteal LNs harvested 8 h p.i. 10 6 OT-I cells were transferred 12–24 h prior to infection. NP-eGFP (no SIINFEKL) was given at 10 7 pfu; all other infections used NP-S-eGFP (containing SIINFEKL) at the indicated dose. Activation was determined by CD69 expression. MFI is shown on the right. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. c ) OT-I CD8 + T cell activation in (b) but 24 h p.i. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. d ) Quantitation of the percentage of VACV- or MVA-infected cells in either the SCS and IFA region or T cell zone contacted by OT-I CD8 + T cells (n = 37 VACV or 32 MVA; dots indicate individual sections). Bars = mean. Error bars = SEM. e ) Percentage of activated OT-I CD8 + T cells (those with CD69 MFI > 50) in each region of 10 LN sections harvested 8 h after infection with 10 8 pfu VACV. Percentages shown are activated cells/total cells in each region. Dots = individual LN sections. Bars = mean. Error bars = SEM. f ) Percentage of only the activated OT-I CD8 + T cells (as opposed to total cells in (e)) found in each LN region. g ) MIP LN section 8 h p.i. showing OT-I CD8 + T cell activation. B = B cell follicle, SCS = subcapsular sinus. Arrow indicates activated T cell cluster in the T cell zone, magnified on the right. Scalebars = 50 μm, left and 5 μm, right. OT-I CD8 + T cells = red, CD69 = white, VACV-infected cells = green, B220 = blue. h ) MFI of CD69 on all activated cells in each region of the popliteal LN shown in (g). n =197 T cells. Bars = mean. Error bars = SEM. i ) MIP of popliteal LN harvested 8 h post infection with 10 8 pfu VACV (green) without OT-I transfer. Endogenous, polyclonal CD8 + T cells = red. Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. B220 = blue, VACV-infected cells = green. The boxed area is magnified in (j). scalebars = 100 μm j ) Higher magnification MIP image of (i) showing both the SCS&IFA (labeled SCS) and T cell zone (T). Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. scalebars = 50 μm. f,h,i,j) 6 LNs from 3 experiments were analyzed. g) 10 LNs from 5 experiments were analyzed. Statistics = unpaired two-tailed t test.

Article Snippet: Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following Abs: CD11b (clone M1/70, eBioscience, Lot#E022424, E15525–01, 4319572, or B259438); CD11c (clone N418, eBioscience, Lot#4288340, 4339477, or E029528); CD205 (clone NLDC-145, Biolegend, Lot#B251007); B220 (clone RA3–6B2, eBioscience, Lot#4288340, 4339477, or 4306068); CD8α (clone 53–6.7, eBioscience, many different lots); Lyve-1 (clone ALY7, eBioscience, Lot#4291625 or 4311240); ERTR7 (staining a fibroblastic reticular cell antigen and used to identify the LN stromal network including conduits, blood vessels and lymphatic sinuses; rat monoclonal, Abcam (cat#51824); CD69 (polyclonal goat, R&D Systems, Cat#AF2386, Lot 0117021 or 0107081); anti-GFP (polyclonal, eBioscience, Lot#E051981634); anti-vaccinia virus (Accurate Chemical Group, Lot#J2368); anti-NS2B (Genetex, Lot#42508); and/or Lyve-1 (clone ALY7, eBioscience, Lot#4291625, 4311240 or 1978147).

Techniques: Infection, Activation Assay, Flow Cytometry, Expressing, Quantitation Assay, Labeling, Two Tailed Test

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Maximum intensity projection (MIPs) of sections from two different popliteal LNs (left and right) harvested 8 h after footpad injection of 10 6 pfu of VACV. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue. Arrows indicate the location of VACV-infected paracortical cell. scalebars = 100 μm. Higher magnification images are shown on the right; lower panels lack the blue channel for clarity. Scalebars = 50 μm. Results are representative of 10 LNs from 3 experiments. b ) MIP of a section of a popliteal LN harvested 8 h after footpad injection of 10 6 pfu of VACV. Prior to infection, 10 6 OT-I CD8 + T cells were transferred (pink). ERTR7 = red, B220 = blue, CD69 = white, VACV-infected cells = green. Arrows indicate two areas of T cell activation that are shown in higher magnification insets on the left. Scalebar = 200 μm. Top panels of insets show merge; bottom panels do not show OT-I signal (pink) in order to reveal CD69 staining more clearly. Scalebars = 20 μm. Results are representative of 10 LNs from 3 experiments. c ) MIP of a section of a popliteal LN harvested 8 h after footpad infection with 10 6 pfu of VACV showing an infected paracortical CD205 + DC. Scalebar = 200 μm. Higher magnification images are shown in the insets showing: top left) merge; top right) conduits + VACV-infected cells; lower left) conduits + VACV-infected cells + CD11c + CD11b; lower right) conduits + VACV-infected cells + CD205. Scalebar = 20 μm. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue, CD11c = yellow, CD205 = purple, and CD11b = brown. Results are representative of 10 LNs from 3 experiments.

Article Snippet: Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following Abs: CD11b (clone M1/70, eBioscience, Lot#E022424, E15525–01, 4319572, or B259438); CD11c (clone N418, eBioscience, Lot#4288340, 4339477, or E029528); CD205 (clone NLDC-145, Biolegend, Lot#B251007); B220 (clone RA3–6B2, eBioscience, Lot#4288340, 4339477, or 4306068); CD8α (clone 53–6.7, eBioscience, many different lots); Lyve-1 (clone ALY7, eBioscience, Lot#4291625 or 4311240); ERTR7 (staining a fibroblastic reticular cell antigen and used to identify the LN stromal network including conduits, blood vessels and lymphatic sinuses; rat monoclonal, Abcam (cat#51824); CD69 (polyclonal goat, R&D Systems, Cat#AF2386, Lot 0117021 or 0107081); anti-GFP (polyclonal, eBioscience, Lot#E051981634); anti-vaccinia virus (Accurate Chemical Group, Lot#J2368); anti-NS2B (Genetex, Lot#42508); and/or Lyve-1 (clone ALY7, eBioscience, Lot#4291625, 4311240 or 1978147).

Techniques: Injection, Infection, Activation Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Mel92.1 and MP41 cells were treated with DMSO, UNC0642 (1 μM), or A366 (1 μM) for 48 h and analyzed for the indicated proteins using immunoblotting. ACTINB was used as a loading control. ( B ) Mel92.1 and MP41 cells expressing either nonspecific (NS) shRNA or TGF-β1 shRNAs were analyzed for the indicated proteins using immunoblotting. ACTINB was used as a loading control. ( C ) Mel92.1 and MP41 cells expressing either an empty vector or V5-tagged TGF-β1 ORF were treated with DMSO or UNC0642 (1 μM) for 48 h and analyzed for the indicated protein using immunoblotting. ACTINB was used as a loading control. ( D ) Mel92.1 and MP41 cells expressing either NS shRNA, ULBP3 shRNAs, or MICB shRNAs were analyzed for the indicated proteins using immunoblotting. ACTINB was used as a loading control. ( E ) Mel92.1 and MP41 cells expressing either NS shRNA or MICB shRNAs were treated with DMSO or EHMT2 inhibitors UNC0642 (1 μM) for 48 h and were analyzed for NK cell-mediated cytotoxicity using an LDH-based cytotoxicity assay. Relative NK cell-mediated cytotoxicity under the indicated conditions are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( F ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h followed by treated NK cells were used for NK cell-mediated cytotoxicity assay against Mel92.1 Cells. Relative NK cell-mediated cytotoxicity under indicated conditions are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( G ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and were analyzed for IFN-γ mRNA levels using RT-qPCR. Relative IFN-γ mRNA levels are presented under the indicated conditions. ACTINB was used for normalization. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( H ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for IFN-γ protein levels using ELISA. IFN-γ protein levels are presented under the indicated conditions. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( I ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for EOMES and T-Bet protein levels using immunoblotting. ACTINB was used as a loading control. ( J ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for NCR3 and NKG2D protein using immunoblotting. ACTINB was used as a loading control. ( K ) NK92MI cells were treated with TGF-β1 (10 ng/ml) for 24 h and analyzed for CD69, CD49a, CD103, CD56, and CXCR6 proteins using immunoblotting. ACTINB was used as a loading control. All quantitative data are shown as the mean ± SEM. .

Article Snippet: CD69 (Diluted at 1:1000 for WB) , Cell Signaling Technology , 39275 RRID: NA.

Techniques: Western Blot, Control, Expressing, shRNA, Plasmid Preparation, LDH Cytotoxicity Assay, Two Tailed Test, Cytotoxicity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hamster monoclonal anti-mouse CD69 - 143Nd , Fluidigm , Cat#3143004B; RRID:AB_2827881.

Techniques: Purification, Recombinant, Activation Assay, SYBR Green Assay, Reverse Transcription, Detection Assay, Enzyme-linked Immunosorbent Assay, Selection, Software

Journal: Cell

Article Title: Complement activation induces excessive T cell cytotoxicity in severe COVID-19

doi: 10.1016/j.cell.2021.12.040

Figure Lengend Snippet: Increased degranulation and cytotoxic potential of T cells from severe COVID-19 (A) Box and whisker (5–95 percentile) plots of SARS-CoV-2-specific IgG and IgA antibody levels detected in serum samples from mild (n = 15) and severe (n = 17) COVID-19 patients collected between day 10 and 14 post symptom onset. Wilcoxon test ∗∗ p < 0.01. (B) Linear regression analysis of TFH cell proportions (CyTOF cluster 7) determined in samples collected from mild (n = 8) and severe (n = 11) COVID-19 (cohort 1) during day 5 and 14 post-symptom onset and SARS-CoV-2-specific IgG and IgA serum levels. (C) Box and whisker (min − max) plots summarizing the intracellular granzyme B expression (unstimulated) of CD8 + T cells from PBMCs of mild (n = 21) and severe (n = 28) COVID-19 patients as well as controls (n = 21). Kruskal-Wallis & post hoc Dunn’s multiple comparison test ∗ p < 0.05, ∗∗∗ p < 0.001. (D) Box and whisker (5–95 percentile) plots summarizing the degranulation capacity of CD8 + T cells from PBMCs of mild (n = 21) and severe (n = 28) COVID-19 patients as well as controls (n = 20) defined by their increase of cell surface CD107a expression upon stimulation with anti-CD16 antibody-coated or isotype-coated beads. Multiple Mann-Whitney test ∗ p < 0.05, ∗∗∗ p < 0.001. (E) Scatter plot of the degranulation capacity from PBMCs of severe COVID-19 patients (n = 12) upon stimulation with spike-protein-coated beads pre-incubated with control serum, with spike-protein-coated beads pre-incubated with COVID-19 serum or with anti-CD16 antibody-coated beads. Kruskal-Wallis and post hoc Dunn’s multiple comparison test ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (F) Enrichment of CD137 + CD69 + cells in activated CD16 − (CD38 + CD16 − ) and activated CD16 + (CD38 + CD16 + ) over the total CD8 + T cell compartment upon restimulation of PBMC samples from mild (n = 5) and severe (n = 7) COVID-19 patients with a SARS-CoV-2 peptide pool. Enrichment was calculated by dividing the proportions of CD137 + CD69 + T cells in non-activated CD16 − (CD38 + CD16 − ) and activated CD16 + (CD38 + CD16 + ) by the proportions of total CD8 + T cells. Friedmann test & post hoc Dunn’s multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01. (G) Box and whisker (5–95 percentile) plots summarizing the normalized release of CXCL8 and CCL2 by primary lung endothelial cells co-cultured with CD8 + T cells enriched from PBMCs of mild (n = 6) and severe (n = 5) COVID-19 patients as well as non-infected controls (n = 5) upon stimulation with ConA or anti-CD16 antibody-coated beads. Wilcoxon test ∗ p < 0.05. (H) Endothelial cell resistance upon stimulation with ConA alone (n = 5) or additional co-culture with CD8 + T cells enriched from PBMCs of mild (n = 6) and severe (n = 5) COVID-19 patients as well as non-infected controls (n = 5). Kruskal-Wallis test ∗ p < 0.05. (I) Representative immunofluorescence staining of CD3 (green) and CD16 (red) in autopsy lung tissues of patients without lung pathology, with COVID-19, ARDS, or influenza pneumonia. (J) Quantification of CD3/CD16 double-positive T lymphocytes per mm 2 in the autopsy cohorts of deceased patients without lung pathology (n = 4) compared with COVID-19 (n = 13), ARDS (n = 8) and influenza pneumonia (n = 6). The COVID-19 cohort was separated into early stage (death after 7–14 days after first symptoms), mid stage (15–30 days after symptom onset), and late stage (>35 days). One-way ANOVA; ∗∗ , p < 0.01.

Article Snippet: CD69 162Dy (FN50) , Fluidigm , Cat# 3162001B; RRID: N/A.

Techniques: Whisker Assay, Expressing, Comparison, MANN-WHITNEY, Incubation, Control, Cell Culture, Infection, Co-Culture Assay, Immunofluorescence, Staining

Journal: Cell

Article Title: Complement activation induces excessive T cell cytotoxicity in severe COVID-19

doi: 10.1016/j.cell.2021.12.040

Figure Lengend Snippet: Representative plots of gating strategy, related to Figure 3 F (A) Gating of CD137 + CD69 + of CD8 + CD16 + CD38 + T cells, also discriminationg between CD8 low and CD8 high cells, as shown in Figure 3 F. Both, CD8 low & CD8 high expressing T cells from patients with severe COVID-19 contain high proportions of CD16 + CD38 + cells.

Article Snippet: CD69 162Dy (FN50) , Fluidigm , Cat# 3162001B; RRID: N/A.

Techniques: Expressing

Journal: Cell

Article Title: Complement activation induces excessive T cell cytotoxicity in severe COVID-19

doi: 10.1016/j.cell.2021.12.040

Figure Lengend Snippet:

Article Snippet: CD69 162Dy (FN50) , Fluidigm , Cat# 3162001B; RRID: N/A.

Techniques: Purification, Functional Assay, Blocking Assay, Recombinant, Lysis, Cell Isolation, Staining, Positive Control, Virus, Software, Derivative Assay